Toward personalized therapy for chronic lymphocytic

Bedside to Bench Report

Bedside to Bench Report

Cancer Biology & Therapy 14:1, 1–7; January 2013; © 2013 Landes Bioscience

Toward personalized therapy for chronic lymphocytic leukemia

Małgorzata Rogalińska,1,† Ida Franiak-Pietryga,2,3,† Jerzy Z. Błoński,2,3 Paweł Góralski,4 Henryk Maciejewski,5 Agnieszka Janus,2 Paweł Robak,6 Marek Mirowski,7 Henryk Piekarski,4 Tadeusz Robak2 and Zofia M. Kiliańska1,* Department of Cytobiochemistry; University of Lodz; Lodz, Poland; 2Department of Hematology; Medical University of Lodz; Lodz, Poland; 3Laboratory of Clinical and Transplant Immunology and Genetics; Copernicus Memorial Hospital; Lodz, Poland; 4Department of Physical Chemistry; University of Lodz; Lodz, Poland; 5 Institute of Computer Engineering, Control and Robotics; Wroclaw University of Technology; Wroclaw, Poland; 6Department of Experimental Hematology; Medical University of Lodz; Lodz, Poland; 7Department of Pharmaceutical Biochemistry; Laboratory of Molecular Biology and Pharmacogenomics; Medical University of Lodz; Lodz, Poland 1

These authors contributed equally to this work.

†

Keywords: apoptosis-related genes/proteins, clinical response, CLL, DSC, immunochemotherapy, gene expression, qPCR cDNA microarray Abbreviations: CLL, chronic lymphocytic leukemia; DSC, differential scanning calorimetry; RCC, rituximab + cladribine + cyclophosphamide; RCM, rituximab + cladribine + mafosfamide; PBMCs, peripheral blood mononuclear cells; CR, complete response/responder; NR, non-response/non-responder; GEP, gene expression profile

The differences in clinical course of chronic lymphocytic leukemia could have an impact on variations in a patient’s response to therapy. Our published results revealed that thermal transition (95 ± 5°C) in differential scanning calorimetry profiles appear to be characteristic for the advanced stage of CLL. Moreover, a decrease/loss of this transition in nuclei from leukemic cells exposed to drugs ex vivo could indicate their diverse efficacy. It seems that the lack of changes in thermal profile could predict patient’s drug resistance. In this study, we demonstrate the results obtained after drug treatment of leukemic cells by calorimetry, apoptosis-related parameters involved in expression of genes using cDNA microarray and western blot. These data were compared with the patients’ clinical parameters before and after RCC therapy (rituximab + cladribine + cyclophosphamide). The complementary analysis of studied cases with opposite clinical response (CR or NR) revealed a strong relationship between clinical data, differences in thermal scans and apoptosis-related gene expression. We quantified expression of eight of apoptosis-related 89 genes, i.e., NOXA, PUMA, APAF1, ESRRBL1, CASP3, BCL2, BCL2A1 and MCL1. Particular differences in NOXA and BCL2 expression were revealed. NOXA expression in cells of patients who achieved a complete response to RCC therapy was 0.44 times higher in comparison to control ones. Interestingly, in the case of patients who did not respond to immunotherapy, NOXA expression was highly downregulated (RQ = 4.39) as compared with untreated cells. These results were confirmed by distinct cell viability, protein expression as well as by differences in calorimetry profiles.

Introduction Despite the growing list of chronic lymphocytic leukemia (CLL) treatment options, this hematological neoplasm still remains incurable and highly heterogeneous.1 The reason for this appears to be the variability in the clinical course of disease, which could originate from aberrations on a genetic level.2,3 Moreover, the simultaneous coexistence of two populations of CLL cells—quiescent in peripheral blood and highly proliferative in patient’s bone marrow germinal centers, requires therapy effective for both resting and cycling CLL cell populations.4-6 Over the past 20 years, an improvement in CLL treatment has occurred by

application of antibiotics, corticosteroids, purine analogs and monoclonal antibodies.7-11 The combined therapy of purine analogs with alkylator significantly increased patients’ response to administered therapy.8,10,11 There are a large number of new anticancer compounds currently undergoing preclinical or clinical tests.12 Some of them reflect their action toward uncycling and cycling cell populations.6,7,12 Despite the increasing number of clinical diagnostic parameters and overall improvement of patients’ response to therapy, there are still patients who display a poor reaction to administered drugs.13,14 The results revealed that in the group of CLL patients treated previously with chlorambucil or fludarabine, an

*Correspondence to: Zofia M. Kiliańska; Email: [email protected] Submitted: 04/20/12; Revised: 09/24/12; Accepted: 10/22/12 http://dx.doi.org/10.4161/cbt.22623 www.landesbioscience.com

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DSC and cDNA microarray assessment of two cases

3°C are consistent with our previously reported data,18 and reveal their predictive value for clinical response for CLL patients much earlier, after ex vivo testing of their PBMC sensitivity to planned treatment options. The differences in studied gene expression, before and after treatment, were considered significant only if RQ values was > 2 or < -2 (indicative of upregulation or downregulation, respectively). The heatmap shows the hierarchical clustering of 89 apoptosis-related genes (Fig. 2, left). The spectacular differences in NOXA and BCL2 expression levels were detected (Table 2). In the PBMCs of patient PZ, NOXA expression was 1.28 times higher in comparison to control ones (before treatment). However, the expression of MCL1 about four times lower was detected in the case of patient PZ, in comparison to the related its expression for RJ (RQ = 8.60). Non-responder (NR). Patient (RJ), who did not respond to therapy (NR), reflected unfavorable unmutated status of IgHV (Table 1). After PBMCs exposure to RCM, the moderate decrease of PBMC viability, i.e., 69.5%, in comparison to control ones, 93.1% (Fig. 1A), occurred. Concomitantly, a slight decline of thermal transition at about 95 ± 3°C in nuclei from PBMCs exposed to RCM was observed (Fig. 1B). Western blot analysis of lysate proteins revealed the dropping of Mcl-1 and Bcl-2 expression (Fig. 1C). In the same blot, a high expression of uncleaved procaspase-3 was noticed. Moreover, a very low basal expression of Noxa in the lysates of untreated CLL cells that diminished in leukemic cells exposed to drugs was found. NOXA expression level was highly downregulated (RQ = 4.39) as compared with untreated cells (Table 2). The similar trend was seen for antiapoptotic genes BCL2, BCL2A1 and proapoptotic ESRRBL1. The complementary studies conducted ex vivo for patient RJ indicated its lower reactivity following the treatment in vivo in comparison with patient PZ. Interestingly, we have noticed only slight changes in gene expression of CASP3, PUMA and APAF1 in the studied PBMC samples of both patients.

Results

Discussion

Complete responder (CR). CLL cells from blood of patient PZ who achieved CR displayed mutated status of IgHV (97.57%). After immunochemotherapy, the complete regression of lymph nodes and lack in spleen and liver enlargement was observed (Table 1). The strong decrease of patient PZ’s cell viability within leukemic cells exposed to RCM (32.7%) in comparison to control ones (76.6%) was seen (Fig. 1A). Importantly, in thermal scans of nuclei from PBMCs incubated for 48 h with RCM, a strong decline of thermal transition at about 95° ± 5°C, in contrast to control ones, occurred (Fig. 1B). It should also be underlined that in the immunoblots of drug-treated cell lysates, the dropping of antiapoptotic Mcl-1 and Bcl-2 protein expression and slight proteolysis of procaspase-3 detected with appropriate antibodies were noticed. Simultaneously, an elevated Noxa expression was also seen in the control, untreated as well as exposed to RCM leukemic cells (Fig. 1C). The patient’s overall reactivity manifested by the changes of parameters related to apoptosis as well as the observation of strong decreases in thermal transition at 95 ±

The integration of pro-survival microenvironmental signals and variations in the intrinsic apoptotic machinery may contribute to CLL cell resistance to apoptosis induction as well as reaching insufficient efficacy of anticancer therapy.19 Moreover, the remarkable heterogeneity in clinical course of this disease, as well as distinct patients’ response to therapy, still revealed a serious problem in CLL treatment.20 To date, knowledge on various proand antiapoptotic gene expression in response to clinically used apoptosis-induced drug(s) in this type of leukemia is scarce. It is plausible that the delicate balance between anti- and proapoptotic protein level could determine the fate of leukemic cells.19,20 The scientists underlined the special importance of Mcl-1 as well as Bcl-2 expression levels for inhibition of apoptosis in CLL cells related to their binding potential to proapoptotic proteins, thereby preventing mitochondrial outer membrane potential obstruction due to apoptosis inhibition.21 Studies regarding the clinical efficacy of different therapeutic options used for CLL treatment have already been performed;

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increase in resistance to a second course of drug administration occurred.15 Therefore, there is a need to tailor therapy for patients as a strategy to avoid ineffective treatment, increase the complete remission rates and to improve patients’ survival rates. Over the last decade, microarray technology has been an innovative tool to study gene expression. This technique provides detailed information about expression levels of simultaneously examined genes and could be helpful in treatment-monitoring of gene expression, particularly related to apoptosis. The limited data concerning apoptotic gene expression changes in CLL cells, mainly obtained from in vivo/ex vivo treatment.16-18 In vitroreported studies were conducted on CLL cells exposed to purine analogs and directed mainly to P53-related genes and drug resistance.16,17 The aim of the present study was to evaluate the ex vivo potential of apoptosis induction in primary tumoral peripheral blood mononuclear cells (PBMCs) by RCM, i.e., rituximab + cladribine + mafosfamide (ex vivo active form of cyclophosphamide) using Vybrant Apoptosis Assay #4, differential scanning calorimetry and western blot (Mcl-1, Bcl-2, procaspase-3, Noxa). The obtained results compared the data from two cured patients receiving opposite clinical responses described as complete responder (CR) or non-responder (NR) after six cycles of immunochemotherapy. Moreover, the expression in vivo of eight of 89 apoptosis-related genes by qPCR microarray in CLL cells from patient’s blood before and 2 wk after first RCC (rituximab + cladribine + cyclophosphamide) cycle of administration was reported. One primary goal in CLL therapy is to reach CR with intensive treatment options. However, there is no clinical evidence that therapy intervention in CLL can improve outcome, even if prognostic parameters are considered. Herewith, we describe the result sets of two patients with newly diagnosed CLL who underwent RCC therapy and reached the distinct clinical response, i.e., complete remission or lack of response.

however, there is no data specifically focused on the possible modification of the gene expression profile (GEP) after the administration of drugs.10 Very recently we have documented the differences in apoptotic GEP among CLL cells exposed to fludarabine or cladribine.22 Hallaert et al.23 reported that Mcl-1 differently binds to proapoptotic proteins, Noxa and Bim, in untreated and seliciclibtreated CLL cells. The cited authors showed that Noxa is required to drive the mitochondrial apoptosis upon this novel CDK inhibitor treatment. Noxa protein remained bound to Mcl-1 in seliciclib exposed CLL cells, while Bim is dissociated. They suggested the sequence of events in seliciclib-treated leukemic cells after Mcl-1 degradation, following by Noxa-dependent Bim displacement, its liberation and finally Bax activation and forming large multimeric

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pores in mitochondria.23 The described findings are consistent with our results reflecting a high-basal Noxa expression in PBMC cells of patient PZ, who reached CR after RCC administration. However, we have observed a low expression of Noxa before drug exposure of patient RJ’s cells and its lack after cell exposure to RCM. As previously identified,24 the appearance of additional thermal transition at about 95 ± 3°C zone in cell nuclei obtained from PBMC samples from patients’ blood could be proposed as a new parameter for the advanced stage of CLL. Interestingly, the decrease of this transition appears to have a prognostic significance and could reflect patients’ potential sensitivity to the treatment in vivo. On the other hand, the lack of differences in thermal scans of nuclei isolated from CLL cells ex vivo exposed to drugs for 48 h may suggest patients’ resistance to therapy.


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Figure 1. Viability of CLL cells (A), differential scanning calorimetry profiles of nuclear fraction (B) and changes in expression of apoptosis-related proteins (Mcl-1, Bcl-2, procaspase-3, Noxa) in PBMC lysates (C), isolated from blood of patients PZ and RJ, incubated for 48 h with and without RCM combination. Immunoblot analysis of PBMCs after their exposure to drug combination were analyzed by alkaline phosphatase method in the presence of appropriate antisera. Protein lysates (50 μg) were loaded into 12.5% polyacrylamide gels; β-actin was used as loading control; Cpex - excess heat capacity.

Materials and Methods Figure 2. Microarray analysis indicating changes in expression of 89 apoptosis-related genes in two CLL samples following immunotherapy (left); boxplots of the DDCT values of samples from patients PZ and RJ (right). The rows and the columns represent individual genes and mRNA samples, respectively. The relative level of gene expression is depicted according to the color scale shown at the bottom of the matrix. The accuracy of reading in the presented experiments is illustrated by boxplots of the signal (signal DDCT ~-logRQ).

It must be underlined that results obtained ex vivo matched with apoptosis-related gene profiling from the same patients administered with RCC in vivo. We have found that ex vivo and in vivo expression of proapoptotic NOXA could be a significant factor related to different patient’s response to the applied therapy. A high expression of Noxa in patient PZ’s cells seems to correlate with its activity in inhibition of antiapoptotic proteins in mitochondrial outer membrane promoting Noxa proapoptotic activity. Accordingly, in PBMCs from blood of patients who achieved CR to RCC therapy, a strong decline in thermal transition at 95 ± 5°C, accompanied by the increase of main transition at about 83 ± 3°C, was observed.24 The differences in thermal

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The peripheral blood samples from two previously untreated progressive CLL patients with leukocytosis 174 and 120 × 109/L, respectively, were tested. The diagnosis of the disease and clinical staging determination were established according to standard clinical, immunological and cytological IWCLL criteria.26 The study was approved by the Local Ethics Committee of the Medical University of Lodz (No. RNN/143/10/KE) and the patients signed a declaration of consent. The patients were of 65 and 54 y old with diagnosed IB and IIB CLL stage, respectively. Table 1 summarizes clinical data for both patients, who indicate no p53 mutation and opposite IgHV mutational status. Blood samples of both patients were collected prior to therapy (experiments ex vivo). The next day, both patients underwent therapy with RCC.27 Clinical response to therapy after six cycles of RCC administration was estimated by NCI Sponsored Working Group criteria.26 The cytogenetic analysis of four of the most frequently detected in CLL aberrations, i.e., del(13)(q14), trisomy 12, del(11)(q22) and del(17)(p13) by fluorescent in situ hybridization (FISH) was evaluated.28,29 PCR amplification of IGHV-IGHD-IGHJ


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scans of nuclear fraction from drug-treated CLL cell samples seems to be a consequence of chromatin remodeling during the apoptosis process. Based on the cell viability results, we perceived the strong correlation between DSC and western blot results (expression/proteolysis level of apoptosis-related proteins) accompanied by high relevance to gene expression profiling data and patients’ clinical monitoring in vivo. The highest differences in NOXA and BCL2 expression on gene and protein expression levels between both examined in vivo and ex vivo patients were revealed. We also matched the results of ex vivo or in vivo experiments with clinical parameters and prognostic markers for these patients. In accordance with accepted hematological clinical parameters for patient PZ’s, who achieved CR to the applied therapy, IGHV-mutated status was confirmed, while patient RJ, who did not reached remission, indicated unfavorable unmutated status of this gene variable region. It is plausible that unmutated IgHV status, and the presence of genomic aberrations (del 11q), reflect link with progressive type of CLL and patients’ shorter survival.25 Our findings suggest the value of ex vivo treatment of CLL cell samples before patient’s drug administration to choose the most effective manner of therapy. In conclusion, special attention should be paid to ex vivo data on CLL cell viability, and some apoptotic events in drug-treated cells together with molecular status of apoptosis-related genes. Additionally, the promising results demonstrate the advantage of differential scanning calorimetry in the evaluation of CLL therapy and reveal that its application could facilitate the choice of effective treatment for individual patients. The validation of presented data by examination of larger groups of CLL patients is needed.

Initials

PZ

RJ

Age/sex

65/M

54/M

Rai

IB

II A

174.63 x 103/μl 13.7 g/dl 193.0 x 103/μl

126.27 x 103/μl 11.1 g/dl 136.0 x 103/μl

3.00 x 103/μl 11.6 g/dl 108 x 103/μl

1.87 x 103/μl 13.5 g/dl 83 x 103/μl

IgHV mutational status

Mutated

Unmutated

Blood count (before treatment) WBC Hb PLT Blood count (assessment of response) WBC Hb PLT CD 38

20%

21%

β2-microglobulin (mg/L)

4.20

8.78

LDH (U/L) (before treatment)

225

260

Cytogenetics

del 11q, del 13q

del 11q

Lymph nodes diameter (before treatment)

Cervical region 2 × 1 cm Axillary region 3 × 4 cm Inquinal region 2 × 2 cm

Cervical region 3 cm Axillary region 3 cm Inquinal region 3 cm

Lymph nodes diameter (during assessment of response)

Complete regression of peripheral and central lymph nodes

Complete regression of peripheral lymph nodes and < 50% regression of central lymph nodes

Spleen and liver (before treatment)

Not enlarged

Spleen enlarged to 8 cm below the left costal margin; Liver enlarged to 4 cm below the left costal margin;

Spleen and liver (during assessment of response)

Not enlarged

Spleen decreased to 2 cm below the left costal margin (CT = 15 cm); Liver enlarged to 3 cm below the left costal margin;

Treatment response*

CR MRD+

NR

The cut-off values for CD38 was 30%. No p53 mutation. *RCC treatment response after 6 cycles.

rearrangements and immunoglobulin sequence analysis were performed as previously described.30 Sequence data were made using the international ImMunoGeneTics (IMGT) information system database and tools: the sequence with a nucleotide identity of 100% as compared with the germline was considered unmutated, while the sequence with an identity of 97.57% as mutated.31 Mononuclear cells from peripheral blood of patients, before RCC administration, were cultured at final density of 3 × 106

cells/ml in RPMI-1640 medium and incubated without (control) or with combination of cladribine (C), mafosfamide (M) and rituximab (R) for 48 h, at concentrations described previously.18,32 Determination of living cell numbers was performed by flow cytometry using Vybrant Apoptosis Assay #4 (Molecular Probes, Inc.). Preparation of whole-cell lysates and nuclear fraction was conducted as previously reported.18 The control and drug-treated CLL cells were lysed. DSC analysis of the nuclear fraction isolated from leukemic PBMCs after incubation for 48

Table 2. Alterations in selected gene expression in two CLL cell samples (patients: PZ and RJ) after 2 wk of RCC therapy illustrated as fold change and ΔΔCt values in reference to control samples (before therapy) PZ

Gene description NOXA (Phorbol-12-myristate-13-acetate-induced protein 1; proapoptotic)

RJ

ΔΔCt

RQ

ΔΔCt

RQ

0.44

1.28

-4.39

161.35

CASP3 (Caspase 3; proapoptotic)

-0.32

2.75

-0.43

3.08

BCL2 (B-cell CLL/lymphoma 2; antiapoptotic)

-0.35

2.84

-3.54

69.60

MCL1 (Myeloid cell leukemia sequence 1; antiapoptotic)

-0.78

4.35

-1.45

8.60

PUMA (P53 upregulated modulator of apoptosis; proapoptotic)

-0.19

2.42

-0.44

3.13

BCL2A1 (BCL2-related protein A1; antiapoptotic)

-0.72

4.11

-1.30

7.36

APAF1 (apoptotic protease activating factor 1; proapoptotic)

-0.76

4.29

-0.41

3.01

ESRRBL1 (IFT57-intraflagellar transport 57 homolog; proapoptotic)

0.47

1.24

-3.01

40.58

ΔΔCt, a value of the difference in expression of a gene between two examined groups (before and after RCC treatment); RQ, fold change (fold difference); RCC, rituximab + cladribine + cyclophosphamide.



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Table 1. Clinical characteristics of patients

References 1. Maddocks KJ, Lin TS. Update in the management of chronic lymphocytic leukemia. J Hematol Oncol 2009; 2:29; PMID:19619273; http://dx.doi. org/10.1186/1756-8722-2-29. 2. Ouillette P, Collins R, Shakhan S, Li J, Peres E, Kujawski L, et al. Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia. Blood 2011; 118:3051-61; PMID:21795749; http:// dx.doi.org/10.1182/blood-2010-12-327858. 3. Zenz T, Mertens D, Döhner H, Stilgenbauer S. Importance of genetics in chronic lymphocytic leukemia. Blood Rev 2011; 25:131-7; PMID:21435757; http://dx.doi.org/10.1016/j.blre.2011.02.002. 4. Reed JC, Pellecchia M. Apoptosis-based therapies for hematologic malignancies. Blood 2005; 106:408-18; PMID:15797997; http://dx.doi.org/10.1182/blood2004-07-2761. 5. Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and malignancy. Nat Rev Immunol 2008; 8:22-33; PMID:18097447; http://dx.doi. org/10.1038/nri2217. 6. Natoni A, Murillo LS, Kliszczak AE, Catherwood MA, Montagnoli A, Samali A, et al. Mechanisms of action of a dual Cdc7/Cdk9 kinase inhibitor against quiescent and proliferating CLL cells. Mol Cancer Ther 2011; 10:1624-34; PMID:21768328; http://dx.doi. org/10.1158/1535-7163.MCT-10-1119. 7. Wesierska-Gadek J, Maurer M, Zulehner N, Komina O. Whether to target single or multiple CDKs for therapy? That is the question. J Cell Physiol 2011; 226:341-9; PMID:20836132; http://dx.doi. org/10.1002/jcp.22426.

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values, defined as the differential gene expression before and after treatment. The complete microarray data are available at Gene Expression Omnibus (accession number GSE33925, samples 17–20). The differences in expression of examined genes between both patients were observed for eight of 89 genes, i.e., NOXA, PUMA, APAF1, ESRRBL1, CASP3, BCL2, BCL2A1 and MCL1. The hierarchical clustering algorithm used was obtained in accordance with the average-linkage clustering method.35 Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

We thank Professor Paolo Ghia (Laboratory of B Cell Neoplasia, Division of Molecular Oncology, San Raffaele Scientific Institute) for sharing his expertise in VHJ mutational status assays. The study was partially supported by Grant No. PBZ/ MNiSW/07/2006/28 from the Polish Ministry of Science and Higher Education, by Grant No. 2011/01/B/NZ4/01702 from Polish National Centre of Science, Poland; by statutory means No. 503/1–093–01/503–01 of the Hematology Department Medical University of Lodz, Poland; by the Foundation for Leukemia Sufferers, Poland and by the Foundation for the Development of Diagnostics and Therapy, Warsaw, Poland.

8. Catovsky D, Richards S, Matutes E, Oscier D, Dyer MJ, Bezares RF, et al.; UK National Cancer Research Institute (NCRI) Haematological Oncology Clinical Studies Group; NCRI Chronic Lymphocytic Leukaemia Working Group. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet 2007; 370:230-9; PMID:17658394; http://dx.doi.org/10.1016/S01406736(07)61125-8. 9. Riaz W, Hernandez-Ilizaliturri FJ, Czuczman MS. Strategies to enhance rituximab anti-tumor activity in the treatment of CD20-positive B-cell neoplasms. Immunol Res 2010; 46:192-205; PMID:19763890; http://dx.doi.org/10.1007/s12026-009-8121-x. 10. Robak T, Jamroziak K, Gora-Tybor J, Stella-Holowiecka B, Konopka L, Ceglarek B, et al. Comparison of cladribine plus cyclophosphamide with fludarabine plus cyclophosphamide as first-line therapy for chronic lymphocytic leukemia: a phase III randomized study by the Polish Adult Leukemia Group (PALG-CLL3 Study). J Clin Oncol 2010; 28:1863-9; PMID:20212251; http://dx.doi.org/10.1200/JCO.2009.25.9630. 11. Flinn IW, Neuberg DS, Grever MR, Dewald GW, Bennett JM, Paietta EM, et al. Phase III trial of fludarabine plus cyclophosphamide compared with fludarabine for patients with previously untreated chronic lymphocytic leukemia: US Intergroup Trial E2997. J Clin Oncol 2007; 25:793-8; PMID:17283364; http:// dx.doi.org/10.1200/JCO.2006.08.0762. 12. Kilianska ZM, Rogalinska M. Potential new agents for chronic lymphocytic leukemia treatment. Anticancer Agents Med Chem 2010; 10:666-82; PMID:21235440. 13. Motta M, Wierda WG, Ferrajoli A. Chronic lymphocytic leukemia: treatment options for patients with refractory disease. Cancer 2009; 115:383041; PMID:19536902; http://dx.doi.org/10.1002/ cncr.24479.


14. Wierda WG, Kipps TJ, Mayer J, Stilgenbauer S, Williams CD, Hellmann A, et al.; Hx-CD20-406 Study Investigators. Ofatumumab as single-agent CD20 immunotherapy in fludarabine-refractory chronic lymphocytic leukemia. J Clin Oncol 2010; 28:1749-55; PMID:20194866; http://dx.doi. org/10.1200/JCO.2009.25.3187. 15. Bosanquet AG, Richards SM, Wade R, Else M, Matutes E, Dyer MJ, et al. Drug cross-resistance and therapyinduced resistance in chronic lymphocytic leukaemia by an enhanced method of individualised tumour response testing. Br J Haematol 2009; 146:384-95; PMID:19552723; http://dx.doi.org/10.1111/j.13652141.2009.07741.x. 16. Plate JM, Petersen KS, Buckingham L, Shahidi H, Schofield CM. Gene expression in chronic lymphocytic leukemia B cells and changes during induction of apoptosis. Exp Hematol 2000; 28:1214-24; PMID:11063869; http://dx.doi.org/10.1016/S0301472X(00)00536-1. 17. Alvi AJ, Austen B, Weston VJ, Fegan C, MacCallum D, Gianella-Borradori A, et al. A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down-regulation of genes involved in transcription regulation and survival. Blood 2005; 105:4484-91; PMID:15692065; http://dx.doi.org/10.1182/blood-2004-07-2713. 18. Rogalinska M, Goralski P, Wozniak K, Bednarek JD, Blonski JZ, Robak T, et al. Calorimetric study as a potential test for choosing treatment of B-cell chronic lymphocytic leukemia. Leuk Res 2009; 33:308-14; PMID:18676014; http://dx.doi.org/10.1016/j.leukres.2008.06.032. 19. Longo PG, Laurenti L, Gobessi S, Sica S, Leone G, Efremov DG. The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood 2008; 111:846-55; PMID:17928528; http://dx.doi.org/10.1182/blood2007-05-089037.

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h with or without anticancer agents was made as described previously.18 Nuclear fraction samples were transferred into steel pans. Calorimetric studies were performed on a Seatram TG-DSC 111 calorimeter from 20–120°C at a scanning rate - R (K/s) of 5 K/min. The analyzed parameter heat flow - HF (J/s) was used to excess heat capacity - Cpex estimation, according to formula: Cpex = HF/R × m; where m is the DNA content (g) in the probe. Protein expression (Mcl-1, Bcl-2, procaspase-3 and Noxa) were analyzed using western blot technique as reported previously.18,33 Total RNA was extracted from cells using RiboPure Blood Kit (Ambion) according to the manufacturer’s instructions. CDNA (cDNA) was synthesized from total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) by priming with random hexamers (0.5 μg of RNA was transcribed to cDNA). Gene expression profiling was carried on with the 384 TaqMan® Low Density Array (Human Apoptosis Panel) (Applied Biosystems) for PCR analysis of 92 gene transcripts (89 examined, three housekeeping genes). cDNA samples were subjected to Real-Time Quantitative PCR (RQ-PCR) in duplicate in the TaqMan® 7900HT Sequence Detection System (Applied Biosystems). The relative expression of each gene was quantified by the comparative cycle threshold (Ct) method (ΔΔCt, DDCT), using 18S as an endogenous control.34 The difference in expression of genes was considered as the fold change


26. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al.; International Workshop on Chronic Lymphocytic Leukemia. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008; 111:5446-56; PMID:18216293; http:// dx.doi.org/10.1182/blood-2007-06-093906. 27. Robak T, Smolewski P, Cebula B, GrzybowskaIzydorczyk O, Błoński JZ. Rituximab plus cladribine with or without cyclophosphamide in patients with relapsed or refractory chronic lymphocytic leukemia. Eur J Haematol 2007; 79:107-13; PMID:17635235; http://dx.doi.org/10.1111/j.1600-0609.2007.00889.x. 28. Döhner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, Bullinger L, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000; 343:1910-6; PMID:11136261; http:// dx.doi.org/10.1056/NEJM200012283432602. 29. Méhes G. Chromosome abnormalities with prognostic impact in B-cell chronic lymphocytic leukemia. Pathol Oncol Res 2005; 11:205-10; PMID:16388316; http:// dx.doi.org/10.1007/BF02893852. 30. Ghia P, Stamatopoulos K, Belessi C, Moreno C, Stella S, Guida G, et al. Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: the lesson of the IGHV3-21 gene. Blood 2005; 105:1678-85; PMID:15466924; http://dx.doi.org/10.1182/blood-2004-07-2606.


31. Brochet X, Lefranc MP, Giudicelli V. IMGT/VQUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res 2008; 36(Web Server issue):W503-8; PMID:18503082; http://dx.doi. org/10.1093/nar/gkn316. 32. Furlan A, Villanova F, Pietrogrande F, Celadin M, Sanzari M, Vianello F. Low-dose fludarabine increases rituximab cytotoxicity in B-CLL cells by triggering caspases activation in vitro. Leuk Lymphoma 2010; 51:107-13; PMID:20001234; http://dx.doi. org/10.3109/10428190903349688. 33. Rogalińska M, Błoński JZ, Komina O, Góralski P, Zołnierczyk JD, Piekarski H, et al. R-roscovitine (Seliciclib) affects CLL cells more strongly than combinations of fludarabine or cladribine with cyclophosphamide: Inhibition of CDK7 sensitizes leukemic cells to caspase-dependent apoptosis. J Cell Biochem 2010; 109:217-35; PMID:19911397; http://dx.doi. org/10.1002/jcb.22400. 34. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402-8; PMID:11846609; http://dx.doi. org/10.1006/meth.2001.1262. 35. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 1998; 95:148638; PMID:9843981; http://dx.doi.org/10.1073/ pnas.95.25.14863.

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20. Chen R, Plunkett W. Strategy to induce apoptosis and circumvent resistance in chronic lymphocytic leukaemia. Best Pract Res Clin Haematol 2010; 23:15566; PMID:20620979; http://dx.doi.org/10.1016/j. beha.2010.01.003. 21. Saxena A, Viswanathan S, Moshynska O, Tandon P, Sankaran K, Sheridan DP. Mcl-1 and Bcl-2/Bax ratio are associated with treatment response but not with Rai stage in B-cell chronic lymphocytic leukemia. Am J Hematol 2004; 75:22-33; PMID:14695629; http:// dx.doi.org/10.1002/ajh.10453. 22. Franiak-Pietryga I, Sałagacka A, Maciejewski H, Błoński JZ, Borowiec M, Mirowski M, et al. Apoptotic gene expression under influence of fludarabine and cladribine in chronic lymphocytic leukemia-microarray study. Pharmacol Rep 2012; 64:412-20; PMID:22661193. 23. Hallaert DY, Spijker R, Jak M, Derks IA, Alves NL, Wensveen FM, et al. Crosstalk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual exhaustion of Bcl-2 protection. Cell Death Differ 2007; 14:1958-67; PMID:17703234; http://dx.doi.org/10.1038/sj.cdd.4402211. 24. Rogalinska M, Góralski P, Kobylinska A, Błonski JZ, Hanausek M, Walaszek Z, et al. Changes in leukemic cell nuclei revealed by differential scanning calorimetry. Leuk Lymphoma 2005; 46:121-8; PMID:15621789; http://dx.doi.org/10.1080/10428190412331283170. 25. Kröber A, Seiler T, Benner A, Bullinger L, Brückle E, Lichter P, et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002; 100:14106; PMID:12149225.